Rapid detection method for multi-species-derived components in dairy products

Abstract

The invention discloses a rapid detection method for multi-species-derived components in dairy products, which is based on a micro-fluidic chip detector, adopts an LAMP nucleic acid isothermal amplification technology, combines a micro-fluidic chip technology, takes the micro-fluidic chip detector as a technical platform, and can systematically serve on-site rapid screening of five species-derived components in dairy products. The detection technology provided by the invention can provide a new technical support for identification of adulterated components of the dairy product, and realizes rapid, accurate, easy-to-operate and low-cost detection of five target source components in the dairy product.

Description

Technical Field

[0002] The invention relates to the technical field of dairy product detection, in particular to a method for rapid detection of components of multiple species origin in dairy products. Background Art

[0003] Nowadays, many species identification detection methods for dairy products have been reported at home and abroad. The traditional protein-based detection methods are mainly divided into: Kjeldahl nitrogen determination, electrophoresis, enzyme-linked immunosorbent assay, chromatography, and spectroscopy. These methods have some shortcomings: 1) poor repeatability; 2) a large amount of organic reagents are required; 3) the operation is complicated and takes a long time; 4) during the processing of dairy products, the structure of protein will be denatured to a certain extent due to heating, which will affect the test results. There are currently two problems in the identification of species adulteration of dairy products: one is the lack of on-site rapid detection technology. The shelf life of dairy products is short, and the existing laboratory tests generally take 2-3 days to produce results, which cannot meet the actual requirements. Therefore, there is an urgent need for on-site rapid identification methods; the second is the lack of multiple adulteration identification technology. Dairy products are mostly liquid milk or made of liquid milk, which can be adulterated and mixed, and multiple tests are required to distinguish the adulterated components. After the emergence of molecular amplification and detection technology based on microfluidics, the complexity of operation requirements has been effectively reduced, making rapid, specific and sensitive on-site detection technology possible.

[0004] The applicant disclosed a microfluidic chip detector in the utility model patent with publication number CN216696351U. Based on this instrument, it is of great significance to develop a rapid detection method for multi-species derived components in dairy products. Summary of the invention

[0005] The present invention aims to at least partially overcome the above technical problems and / or other potential problems existing in the prior art: to provide a method for rapid detection of components of multiple species origin in dairy products.

[0006] The technical solution of the present invention is as follows: A rapid detection method for multi-species derived components in dairy products, comprising the following steps: 1) First, we searched for target sequences from five species, namely cattle, sheep, horses, camels and soybeans, through NCBI GenBank, and designed multiple pairs of specific primers for the conserved gene fragments of the target sequences. The designed primers were tested and verified using clinical samples, and the optimal primers were determined; 2) After the optimal primers determined in step 1) are fixed at the corresponding positions of the microfluidic chip of the microfluidic chip detector, the dairy sample to be tested is added to the sample injection port of the microfluidic chip. The microfluidic chip detector uses centrifugal force to drive the sample into the reaction hole of the microfluidic chip to mix with the reaction solution. The microfluidic chip detector then performs constant temperature amplification, and the amplification product is effectively combined with the fluorescent substance. The fluorescence signal is captured in real time by the fluorescence detector. According to the appearance time, intensity and position of the real-time fluorescence signal, it is judged whether the sample contains the above five species.

[0007] As an optimization, the composition of the reaction solution in step 2) is as follows: 20 mM Tris-HCl 0.2 μL 40 mM KCl 6.8 μL 100 mM (NH4)2SO4 1.8 μL 80 mM MgSO4 1.8 μL 1% Tween-20 1.8 μL 28 mM dNTPs 0.9 μL 8000U / mL Bst enzyme 1.8 μL 1 mM SYBRGREEN fluorescent dye 0.9 μL 4.0×10 -6 mol / L Gold nanoparticles 1.8 μL ddH2O 0.2 μL The beneficial effects of the present invention are as follows: the present invention adopts LAMP nucleic acid constant temperature amplification technology, combines microfluidic chip technology, and uses a microfluidic chip detector as a technical platform, which can systematically serve the on-site rapid screening of five species-derived components in dairy products. The detection technology of the present invention can provide new technical support for the identification of adulterated ingredients in dairy products, and realize rapid, accurate, easy-to-operate, and low-cost detection of five target-derived components in dairy products. DETAILED DESCRIPTION

[0008] The present invention is further described in detail below with specific examples, but the present invention is not limited to the following specific examples. Example

[0009] The instrument used in this embodiment is a microfluidic chip detector disclosed in the utility model patent with publication number CN216696351U. 1) First, we searched for target sequences from five species, namely cattle, sheep, horses, camels and soybeans, through NCBI GenBank, and designed multiple pairs of specific primers for the conserved gene fragments of the target sequences. The designed primers were tested and verified using clinical samples, and the optimal primers were determined; 2) After the optimal primers determined in step 1) are fixed at the corresponding positions of the microfluidic chip of the microfluidic chip detector, the dairy sample to be tested is added to the sample injection port of the microfluidic chip. The microfluidic chip detector uses centrifugal force to drive the sample into the reaction hole of the microfluidic chip to mix with the reaction solution. The microfluidic chip detector then performs constant temperature amplification, and the amplification product is effectively combined with the fluorescent substance. The fluorescence signal is captured in real time by the fluorescence detector. According to the appearance time, intensity and position of the real-time fluorescence signal, it is judged whether the sample contains the above five species.

[0010] As an optimization, the composition of the reaction solution in step 2) is as follows: 20 mM Tris-HCl 0.2 μL 40 mM KCl 6.8 μL 100 mM (NH4)2SO4 1.8 μL 80 mM MgSO4 1.8 μL 1% Tween-20 1.8 μL 28 mM dNTPs 0.9 μL 8000U / mL Bst enzyme 1.8 μL 1 mM SYBRGREEN fluorescent dye 0.9 μL 4.0×10 -6 mol / L Gold nanoparticles 1.8 μL ddH2O 0.2 μL The two sample addition holes of the microfluidic chip of the microfluidic chip detector correspond to the eight detection holes. After the dairy product samples to be detected are placed in the sample addition holes, the sample addition holes are sealed with sealing films and then put on the machine; Reaction system and conditions Serial number Material Name 1 copy (ul) 8 wells 1 Reaction solution 10 2 sample 15 3 Total volume (ul) 25 The temperature was set at 63.5 °C and the reaction time was set at 30 min.

[0011] Microfluidic chip amplification: Since this method uses constant temperature amplification, it does not require the temperature change process of PCR amplification such as denaturation, annealing and extension. The entire reaction process is completed under constant temperature conditions. The amplification program: the temperature is set to 63.5℃, and the reaction time is set to 30min. The running program: the low-speed centrifugation speed is 1600 r / min, the low-speed centrifugation time is 10 sec, the high-speed centrifugation speed is 4600 r / min, and the high-speed centrifugation time is 30 sec.

[0012] Microfluidic chip result judgment: 1. Microfluidic chip detector threshold line setting The threshold line is generally set to 800 (it can be adjusted according to actual conditions, and the setting principle is that the threshold line just exceeds the highest point of the atypical S-type amplification curve, and the Ct value is displayed as 30), and the instrument supporting software automatically analyzes the results.

[0013] 2. Quality Control When the internal reference shows an amplification curve and the Ct value is <30, the experimental results are valid.

[0014] 3. Result determination 3.1 Experimental conditions Positive control: Ct<30, the reaction wells of the positive control and the internal standard reaction wells have obvious typical S-shaped amplification curves.

[0015] Negative control: Ct<30, the negative control reaction wells had no amplification curve, and the internal standard reaction wells had an obvious typical S-shaped amplification curve.

[0016] 3.2 Criteria Positive: Within 30 minutes of reaction time, an obvious amplification curve appears in the project detection well, and the Ct value is <30, and the detection item corresponding to the well is judged to be positive. Negative: Within 30 minutes of reaction time, no obvious amplification curve appears in the project detection well, and the detection item corresponding to the well is judged to be negative.

[0017] The isothermal amplification of the microfluidic chip is performed on the microfluidic chip detector. The instrument will perform real-time fluorescence detection and make judgments based on the effective amplification curve of the fluorescence detection. If there is a standard S-shaped amplification curve in any detection well, the well is judged to be positive, that is, the sample contains the nucleic acid of the corresponding detection well; the detection well without an amplification curve is judged to be negative, that is, the sample does not contain the nucleic acid of the corresponding detection well.

[0018] The above are only examples of the features of the present invention and do not constitute any limitation on the protection scope of the present invention. Any technical solution formed by equivalent exchange or equivalent replacement falls within the protection scope of the present invention.

Claims

1. A rapid detection method for multiple species-derived components in dairy products, characterized in that: The following steps are involved: 1) First, we searched for target sequences from five species, namely cattle, sheep, horses, camels and soybeans, through NCBI GenBank, and designed multiple pairs of specific primers for the conserved gene fragments of the target sequences. The designed primers were tested and verified using clinical samples, and the optimal primers were determined; 2) After the optimal primers determined in step 1) are fixed at the corresponding positions of the microfluidic chip of the microfluidic chip detector, the dairy sample to be tested is added to the sample injection port of the microfluidic chip. The microfluidic chip detector uses centrifugal force to drive the sample into the reaction hole of the microfluidic chip to mix with the reaction solution. The microfluidic chip detector then performs constant temperature amplification, and the amplification product is effectively combined with the fluorescent substance. The fluorescence signal is captured in real time by the fluorescence detector. According to the appearance time, intensity and position of the real-time fluorescence signal, it is judged whether the sample contains the above five species.

2. The rapid detection method for multiple species-derived components in dairy products according to claim 1, characterized in that: The composition of the reaction solution in step 2) is as follows: 20 mM Tris-HCl 0.2 μL 40 mM KCl 6.8 μL 100 mM (NH4)2SO4 1.8 μL 80 mM MgSO4 1.8 μL 1% Tween-20 1.8 μL 28 mM dNTPs 0.9 μL 8000U / mL Bst enzyme 1.8 μL 1 mM SYBRGREEN fluorescent dye 0.9 μL 4.0×10 -6 mol / L Gold nanoparticles 1.8 μL ddHO 0.2 μL.